During its life cycle, the social amoeba Dictyostelium discoideum alternates between a predatory amoeba and a facultative multicellular form. The single-celled amoeba is a well-established model system to study cell-autonomous mechanisms of phagocytosis and defence against intracellular bacterial pathogens, whereas the multicellular forms are arising as models to study the emergence of innate immune defence strategies. Importantly, during evolution, prokaryotes have also evolved their own strategies to resist predation. Considering these complex ecological relationships, we wondered whether D. discoideum cells infected with intracellular pathogenic mycobacteria would be able to undergo their developmental cycle and what would be the fate of the infection. We show that the combination of cellautonomous mechanisms and the organisation into a multicellular organism leads to the efficient multistep-curing of a mycobacteria-infected population, thereby ensuring germ-free spores and progeny. Specifically, using a microfluidic device to trap single infected cells, we revealed that in the first curing phase, individual cells rely on three mechanisms to release intracellular bacteria: exocytic release, ejection and lytic release. The second phase occurs at the collective level, when remaining infected cells are excluded from the forming cell aggregates.

24Phagocytes capture invader microbes within the bactericidal phagosome. Some pathogens subvert killing 25 by damaging and escaping from this compartment. To prevent and fight bacterial escape, cells contain and 26 repair the membrane damage, or finally eliminate the cytosolic escapees. All eukaryotic cells engage highly 27 conserved mechanisms to ensure integrity of membranes in a multitude of physiological and pathological 28 situations, including the Endosomal Sorting Complex Required for Transport (ESCRT) and autophagy 29 machineries. In Dictyostelium discoideum, recruitment of the ESCRT-III protein Snf7/Chmp4/Vps32 and 30the ATPase Vps4 to sites of membrane repair relies on the ESCRT-I component Tsg101 and occurs in 31 absence of Ca 2+ . The ESX-1 dependent membrane perforations produced by the pathogen Mycobacterium 32 marinum separately engage both ESCRT and autophagy. In absence of Tsg101, M. marinum escapes earlier 33 to the cytosol, where it is restricted by xenophagy. We propose that ESCRT has an evolutionary conserved 34 function in containing intracellular pathogens in intact compartments. 35 36 37 38 39 40 41 42 Keywords 43 ESCRT, autophagy, membrane damage, membrane repair, infection, innate immunity, ESX-1, 44 Mycobacterium-containing vacuole, Dictyostelium discoideum, Mycobacterium marinum. 45 46 3 Introduction 47After phagocytic uptake, the closely related pathogenic bacteria Mycobacterium tuberculosis and M. 48 marinum reside in an altered and maturation-arrested phagosome, thereby avoiding its toxic chemical 49 environment 1 , but remaining protected from the cell-autonomous cytosolic defences 2 . This Mycobacterium-50 containing vacuole (MCV) becomes permissive for the bacilli to survive and replicate 3, 4 . However, bacteria 51 access to nutrients is limited. To circumvent this restriction, tubercular mycobacteria damage the MCV and 52 escape to the cytosol. The site of MCV rupture becomes a complex battlefield where various machineries 53 cooperate to repair membrane damage and control cytosolic bacteria. Here, we used the Dictyostelium 54 discoideum-M. marinum system to study the role of Endosomal Sorting Complex Required for Transport 55 (ESCRT) and autophagy in membrane repair during both sterile and pathogen-induced damage. We show 56 that the function of ESCRT-III in membrane repair is evolutionarily conserved, that it contributes to the 57 integrity of the MCV and plays an unrecognised role in cell-autonomous defence. We also provide evidence 58 that the ESCRT-III and autophagy pathways act in parallel to repair endomembrane compartments, but 59 differ in their ability to restrict mycobacteria growth in the cytosol of infected cells. 60 61To access the cytosol, mycobacteria make use of a crucial pathogenicity locus, the Region of 62 Difference 1 (RD1), which encodes the ESX-1 system responsible for the secretion of the membranolytic 63 peptide ESAT-6 5 . The membrane perforations produced by ESAT-6 cause MCV rupture and bacterial 64 escape to the cytosol 3, 6, 7 , a step that precede...